Vaccinia virus (VV) is the live vaccine responsible for successful eradication of smallpox worldwide. This success has led to development of recombinant VV as a vaccine vehicle for other infectious diseases and cancer. However, the development of VV vaccines for use in humans is limited by concerns about safety. Furthermore, the efficacy of VV vaccines has been limited by the pre-existing neutralizing antibodies in people who have been previously vaccinated against smallpox. Thus, there is a need to understand immunological mechanisms underlying the unique potency of VV vaccines, which in turn will help develop safer vaccines that are highly immunogenic but have minimal pathogenic potential. Recent advances in immunology have suggested a crucial role of the innate immune system in shaping adaptive immunity. We have shown that innate immune recognition of VV is mediated by Toll-like receptor 2 (TLR2) and dependent on MyD88 (a common adaptor for TLR signaling) and that the activation of TLR2-MyD88 pathway is critical for adaptive CD8 T cell immunity to VV in vivo. How activation of innate immunity promotes adaptive CD8 T cell responses to VV remains largely unknown. We have found in the preliminary studies that defective TLR signaling on APCs only moderately affected CD8 T cell priming, but not CD8 memory formation in response to VV infection in vivo and that direct TLR2 signaling on CD8 T cells enhanced their proliferation and survival in vitro. The objective of this application is to test the hypothesis that in addition to previously observed TLR-induced APC maturation, direct TLR2 signaling on CD8 T cells intrinsically is also critical for efficient CD8 T cell priming and formation of long- lived memory cells in vivo. Specifically, we will pursue the following two aims: 1) To test if direct TLR2-MyD88 signaling on CD8 T cells is required for their clonal expansion and effector differentiation following VV infection in vivo;2) To define the role of intrinsic TLR2-MyD88 signaling in regulating the formation of long-lived memory CD8 T cells in response to VV infection in vivo. The outcome of these studies will not only shed light on how innate immunity modulates the formation of long-lived protective immunity, but more importantly, lead to the development of new generations of safer, effective vaccines to combat infectious diseases including smallpox, and cancer. PUBLIC HEALTH RELEVANCE: Vaccinia virus (VV) is the live smallpox vaccine responsible for successful eradication of smallpox worldwide. This success has led to development of recombinant VV as a vaccine vehicle for other infectious diseases and cancer. However, the development of VV vaccines for use in humans is limited by concerns about safety and reduced efficacy due to the pre-existing neutralizing antibodies in people who have been previously vaccinated against smallpox. Thus, there is a need to understand immunological mechanisms underlying the unique potency of VV vaccines, which in turn will help develop safer vaccines that are highly immunogenic but have minimal pathogenicpotential. The proposed work is to investigate immunological basis of live VV vaccines in the generation of effective and long-lasting CD8 T cell immunity, which in turn will lead to the development of new generations of safer vaccines to combat infectious diseases including smallpox, and cancer in humans.